Summary of work:The development and maturation of neuronal phenotypes depends upon the factors that regulate the cell sorting, processing and post- translational modifications of cytoskeletal molecules. Among the many cytoskeletal molecules in nerve cells, the neurofilaments (NFs) are exclusively neuronal and serve as phenotypic markers. NFs, neuron specific Class IV intermediate filaments, are the major cytoskeletal element of large axons and together with microtubules, they determine the size and shape of the neuron. The NF-subunit proteins, NF-L, NF-M and NF-H, are extensively phosphorylated, with up to 100 or more potential phosphorylation sites in different domains. After their synthesis in cell bodies they are transiently phosphorylated in head domain sites by various kinases (e.g., PKA, PKC) and are transported in the axon where the hypervariable lys-ser- pro (KSP) repeat sequences in the C- terminal tail domains of NF-M and NF-H are phosphorylated by proline-directed kinases. Phosphorylated tail domains protrude outward from the 10nm filament core and form NF-side arms/ crossbridges, which interact with one another and other cytoskeletal molecules to stabilize the axonal cytoskeleton, increase axon caliber and conduction velocity. During NF processing from cell body to axon terminal, phosphorylation is topographically regulated by a dynamic equilbrium between the activities of kinases and phosphatases within cellular compartments. Though all kinases, phosphatases, regulators and substrates are synthesized in cell bodies, the extensive stable phosphorylation of NFP tail domains occurs primarily in the axon during axonal transport. Inasmuch as NFs require multi-site phosphorylation, it is possible that during NF processing, a sequence of multi-site phosphorylations and dephosphorylations are required before the KSP repeat sites become accessible for axonal phosphorylation. Our focus has been to study NF phosphorylation by asking the following questions: (1) What are the in vivo phosphorylation sites in NFPs? (2) Which kinases and phosphatases are involved in regulating phosphorylation at these sites? and (3) how the activities of these kinases are topographically regulated? We have characterized the in vivo phosphorylated sites in rat and human NF-H and found that almost all are located in the KSP repeats of C-terminal tail domains. The peptide sequences containing these phosphorylated residues are the consensus sequences of proline directed kinases. We have also characterized two major neuronal proline-directed kinases, cdk5 and MAP kinase (Erk1/2) involved in their phosphorylation and identified a phosphatase, PP2A, which dephosphorylates cdk5-phosphorylated sites in NF-H. Unlike other cyclin dependent kinases, cdk5 activity is regulated by neuron specific molecules p35 (35 kDa) (and truncated versions). Cdk5 specifically phosphorylates KSPXK motifs and in the case of rat NF-H, only a small number (10%) of the 52 total KSP repeats. On the other hand, Erk1/ Erk2 phosphorylated the diverse KSPXKX, KSPXXKX, KSPXXXK and KSPXXXXK repeat motifs present in rat NF-M and NF-H with greater efficiency. Moreover, in vivo, we have also shown that a specific inhibitor of MEK (the upstream activator of Erk1/2 in the MAPK cascade) inhibited phosphorylation of NF-M and NF-H and microtubule associated protein (MAP) in primary hippocampal cells in culture.